High expression of the epidermal growth factor receptor (EGFR) in breast carcinoma confers a growth advantage to the tumor cells. The EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 ('Iressa') has clinical activity in a wide range of tumor types, although the mechanism(s) by which it exerts its antitumor activity effects remain unclear. We analyzed the ability of ZD1839 to induce apoptosis and/or inhibition of proliferation in breast carcinoma cell lines, as well any association between this ability and the downregulation activity of MAPK and Akt, two recently proposed markers of ZD1839 activity. Proliferation, survival, and activation of Akt and MAPK were evaluated in six human breast cancer cell lines expressing various levels of EGFR and HER2 and exposed to ZD1839. EGFR and HER2 expression levels were determined using specific monoclonal antibodies and FACS analysis. The effects of ZD1839 were independent of EGFR expression levels, but were influenced by high HER2 expression. ZD1839 significantly reduced the rate of [3H]-thymidine incorporation in the four sensitive cell lines, while apoptosis was also induced in two of these cell lines. No correlation was found between the cytostatic or cytotoxic effects of ZD1839 and its ability to downregulate MAPK and Akt activity in the tumor cell lines. Our data suggest that the antitumor activity of ZD1839 is due to a cytostatic effect, and involves apoptosis induction in a subset of sensitive cells only, and that neither MAPK nor Akt is a reliable marker of ZD1839 activity.

Uncontrolled insertion of gene transfer vectors into the human genome is raising significant safety concerns for their clinical use. The wild-type adeno-associated virus (AAV) can insert its genome at a specific site in human chromosome 19 (AAVS1) through the activity of a specific replicase/integrase protein (Rep) binding both the AAVS1 and the viral inverted terminal repeats (ITRs). AAV-derived vectors, however, do not carry the rep gene and cannot maintain site-specific integration properties. We describe a novel hybrid vector carrying an integration cassette flanked by AAV ITRs and a tightly regulated, drug-inducible Rep expression cassette in the framework of a high-capacity, helper-dependent adenoviral (Ad) vector. Rep-dependent integration of ITR-flanked cassettes of intact size and function was obtained in human primary cells and cell lines in the absence of selection. The majority of integrations were site specific and occurred within a 1000-bp region of the AAVS1. Genome-wide sequencing of integration junctions indicates that nonspecific integrations occurred predominantly in intergenic regions. Site-specific integration was obtained also in vivo, in an AAVS1 transgenic mouse model: upon a single tail vein administration of a nontoxic dose of Ad/AAV vectors, AAVS1-specific integrations were detected and sequenced in DNA obtained from the liver of all animals in which Rep expression was induced by drug treatment. Nonrandom integration of double-stranded DNA can therefore be obtained ex vivo and in vivoby the use of hybrid Ad/AAV vectors, in the absence of toxicity and with efficiency compatible with gene therapy applications.

Fhit phosphorylation proliferation ͉ src ͉ proteasome inhibition

The role of HER2 in predicting response to doxorubicin (DXR) therapy in breast cancer was evaluated in vivo in a series of breast carcinomas from 220 patients with tumors larger than 2.5 cm and treated with 3 cycles of DXR (75 mg/m(2)) as neoadjuvant chemotherapy. Patients with HER2-positive tumors were more frequently responsive to DXR treatment compared with HER2-negative patients (p = 0.05; Mantel-Haenszel Chi(2) = 0.009). Progesterone receptor (PgR) negativity, but not mutated p53, was also associated with response to DXR (p = 0.05; Mantel-Haenszel Chi(2) = 0.004). Further analysis of those correlations using breast carcinoma cell lines characterized for different biologic parameters revealed a trend between HER2 positivity/PgR negativity and greater DXR sensitivity, but the strongest direct correlation was found between the proliferation rate and sensitivity to DXR (r = 0.82, p = 0.00009). Neither p53 nor the DXR target molecule topoisomerase-II-alpha was significantly associated with in vitro sensitivity to DXR. Thus, whereas data showed that the major biologic parameter associated with in vitro response to DXR in breast cancer cells appears to be the tumor proliferation rate, HER2 expression together with PgR negativity may serve as the counterpart of the proliferation marker in predicting the in vivo response to DXR.

The viral infectivity factor (Vif) is essential for HIV-1 infectivity and hence is an ideal target for promising anti-HIV-1/ AIDS gene therapy. We previously demonstrated that F12-Vif mutant inhibits HIV-1 replication in CD4 ؉ T lymphocytes. Despite macrophage relevance to HIV-1 pathogenesis, most gene therapy studies do not investigate macrophages because of their natural resistance to genetic manipulation. Here, we confirm the F12-Vif antiviral activity also in macrophages differentiated in vitro from transduced CD34 ؉ human stem cells (HSCs). Moreover, we identified the 126-to 170-amino-acid region in the Cterminal half of F12-Vif as responsible for its antiviral function. Indeed, Chim3 protein, containing this 45-amino-acid region embedded in a WT-Vif backbone, is as lethal as F12-Vif against HIV-1. Of major relevance, we demonstrated a dual mechanism of action for Chim3. IntroductionAnti-HIV-1 gene therapy is based on the concept that autologous hemopoietic stem cells (HSCs) carrying antiviral transgenes once reinfused into HIV-1 ϩ subjects will naturally expand and differentiate into CD4 ϩ T cells and macrophages, the 2 major HIV-1 cellular hosts. HIV-1-resistant CD4 ϩ T cells and macrophages will then contribute to reconstitute the heavily compromised immune system of AIDS patients. 1 It is manifest that an anti-HIV-1/AIDS gene therapy strategy should affect early step(s) of HIV-1 life cycle, such as viral entry and proviral DNA integration, to be most effective. This will reduce the generation of infected cells from noninfected cells. 1,2 Viral infectivity factor (Vif) is an HIV-1 key protein because it counteracts the action of the cellular anti-HIV-1 restriction factor human APOBEC3G (hA3G) that, in the absence of Vif, has a deadly effect on HIV-1 replication. 3 Vif is therefore an excellent target for the development of new anti-HIV-1/AIDS gene therapy approaches. In this context, we have previously reported that the natural F12-Vif mutant, containing 14 unique amino-acid substitutions, 4 once delivered in CD4 ϩ T lymphocytes by second-generation lentiviral vector (LV), efficiently prevents HIV-1 production. 5 Recently, several Vif functional domains involved in either protein-protein or protein-viral RNA interactions have been identified. [6][7][8][9][10] Of particular interest is the identification of a novel HCCH zinc-coordination motif conserved among primate lentivirus Vifs. [11][12][13] This motif is critical for the interaction of Vif and Cullin5, which is one of the E3 ubiquitin ligase multiprotein complex deputed to hA3G degradation. 14 Gene therapy preclinical studies, aimed at providing safety, feasibility, and efficacy analyses, mainly privilege the use of CD4 ϩ T lymphocytes because these cells are easy to obtain, cultivate, and transduce by viral vectors, and are highly susceptible to HIV-1 infection in vitro. In contrast, primary macrophages are nonproliferating terminally differentiated cells, which are transduced by LVs at efficiency not higher than 30%. [15][16][17][18] Hence, despi...